【摘要】 目的探讨瘦素(Leptin)体外刺激对主动脉瓣膜间质细胞(VICs)成骨分化的作用及其机制。方法胶原酶消化法分离并培养猪主动脉瓣膜间质细胞,采用100 ng/ml Leptin刺激细胞24 h,提取细胞RNA以及蛋白,通过实时荧光定量-聚合酶链反应(qRT-PCR)以及免疫印迹试验(Western blot)分别测定成骨标志物核心结合因子α1(RUNX-2) mRNA及蛋白的表达情况,同时采用Western blot检测核因子(NF)-κB总蛋白及磷酸化NF-κB蛋白(pNF-κB)的表达情况。在Leptin刺激的同时,采用NF-κB特异性抑制剂SN50干预VICs,再次检测RUNX-2的表达情况。结果 Leptin刺激导致Runx-2 mRNA及蛋白表达显著上调,同时p-NF-κB蛋白表达显著增加,而SN50干预后Runx-2蛋白表达显著下调(P均<0. 05)。结论 Leptin可致VICs成骨标志物RUNX-2表达增加,SN50干预可致RUNX-2蛋白表达下调,表明其能够诱导VICs向成骨细胞分化,并且这种作用是通过NF-κB相关信号通路实现的。更多还原

【Abstract】 Objective To investigate the effect of leptin on osteoblastic differentiation of aortic valve interstitial cells(VICs) and its mechanism. Methods Porcine aortic VICs were separated and cultured by collagenase digestion method.After being stimulated with 100 ng/ml leptin for 24 hours,VICs RNA and protein were extracted. Quantitative real-time polymerase chain reaction(qRT-PCR) and Western blot were used to detect the expressions of osteogenic marker core binding factor α1(runt-related transcription factor-2,RUNX-2) mRNA and protein,respectively. Western blot was used to detect the expressions of nuclear factor(NF)-κB total protein(t-NF-κB) and phosphorylated NF-κB protein(p-NF-κB). At the same time as leptin intervention,the VICs were intervened by SN50(NF-κB-specific inhibitor),and then the expression of RUNX-2 protein was detected again. Results RUNX-2 mRNA and protein expressions and p-NF-κB protein expression were significantly up-regulated after leptin stimulation,but RUNX-2 protein expression was significantly downregulated after SN50 intervention(all P < 0. 05). Conclusions Leptin can lead to the increase of osteogenic marker RUNX-2 expression,while SN50 intervention can induce down regulation of RUNX-2 protein expression. It is that leptin can induce VICs differentiation to osteoblast cells,and this effect is achieved by NF-κB-related signal pathway.更多还原